Three distinct areas were studied in the this project in FY2016: 1. A functional immune system is dependent on the maintenance of gene expression and transcription factors play critical roles in regulating gene expression at specific stages of development. The Ikaros transcription factor family is one such family whose expression is indispensable for immune system development and function. We have been studying the role of one member of this family, Helios, in the function of Treg cells. We had previously demonstrated that Helios was selectively expressed by 70-80% of mouse and human Foxp3+ peripheral Tregs and suggested that expression of Helios allowed the differentiation of tTregs from pTregs. Mice with a T cell-specific deletion of Helios were phenotypically normal, but Helios expression is transiently upregulated in T conventional (Tconv cells) following immunization in vivo. Our previous failure to observe a phenotype in mice with a T cell-specific deletion of Helios may have resulted from a deletion of Helios in both Tregs and Tconv cells. To determine the role of Helios in Treg-function, we generated mice with a Treg-specific deletion of Helios by crossing Heliosfl/fl mice to Foxp3yfp-Cre mice. Mice with a Treg-specific deletion of Helios showed signs of a systemic autoimmune disease beginning at 2 months of age. At 6 months of age, the mice exhibited generalized splenomegaly, lymphadenopathy, expansion of Th1 Teff cells, hypergammaglobulinemia, and increased size/numbers of lymphoid follicles and germinal centers. These mice also developed a marked abnormality of lipid metabolism characterized by a complete absence of subcutaneous and visceral fat and markedly enlarged livers with lipoidosis. None of these abnormalities was observed in aged mice with a deletion of Helios in mature T cells or T cell precursors. Helios-deficient Tregs were as efficient as control Tregs in mediating suppression in vitro and in vivo in preventing the induction of IBD. The most prominent abnormality observed in mice with a Treg-specific deletion of Helios was the failure of the deficient Treg to control T follicular helper (Tfh) function. Taken together, our studies indicate that Helios plays a complex role in Treg function and probably in the function of Tconv cells. 2. Tregs are critical to the maintenance of immune homeostasis and tolerance. Treg cells, CD4+Tconv cells, and CD8+ T cells represent heterogeneous populations composed of memory phenotype (MP, CD44hi) and naive phenotype (NP, CD44low) subpopulations. One of the most prominent characteristics of MP Tregs and MP CD4+ and CD8+ T cells is their high degree of cell cycling in vivo (10% proliferate/day). Very little is known about the contribution of TCR signals alone or the interrelationships between TCR and co-stimulatory signals on Treg homeostasis. The major goal of our studies was to examine the effects of cytokines, the microbiome, the effects of CD28 and CTLA-4 interactions with CD80/CD86, and the role of MHC class II interactions on Treg subset homeostasis and to compare these results to the effects of manipulation of these pathways on the homeostasis of NP and MP CD4+ and CD8+ T cell subpopulations. To examine the role of IL-2 in Treg homeostasis, we administered an anti-IL-2 mAb to wild type (WT) mice. Anti-IL-2 had no effect on the absolute numbers of MP Treg or their cycling as analyzed by Ki-67 expression. The numbers and phenotype of NP and MP Treg in WT and germ-free mice were identical indicating that antigens derived from the microbiome do not play a role in driving MP Treg. In contrast, CTLA-4-Ig markedly reduced the cycling and numbers of MP Treg and MP CD4+ T cells, with minimal effect on the NP T cell subpopulations. Blockade of MHC class II-TCR interaction led to selective expansion of MP Treg cells and MP CD4+ and CD8+ T cells that was reversed upon co-treatment with CTLA-4-Ig. Treatment with anti-CTLA-4 antibody altered MP Treg cell and MP CD4+ and CD8+ T cell homeostasis in a manner similar to anti-MHC class II. Treg cell homeostasis is thus controlled by a complex pathway in which CD28 is the primary driver of proliferation, while TCR and CTLA-4 function as the brakes. 3. The factors modulating pTreg induction remain poorly understood. It is likely that the cytokine microenvironment and TGF-beta1 play important roles. One of the early events in T cell activation during their interaction with DC is the amplification of the synthesis of several of the components of the alternative complement pathway including C3a and C5a and their receptors. The engagement of C3aR and C5aR on both T cells and DCs provides a potent co-stimulatory signal to both cell partners. We asked whether inhibition of C3aR and C5aR signaling in both T cells and DCs might promote the induction of iTregs in vitro and pTregs in vivo. We have addressed the role of DAF (CD55) in tolerance induction in vivo by using DAF-/- mice. DAF normally restrains C3a and C5a generation. Oral tolerance was abrogated in DAF -/- mice. We have also demonstrated that Tregs generated in the absence of C3aR/C5aR signaling have greater stability of Foxp3 expression upon antigen exposure and have de-methylated a critical region of the Foxp3 gene (the TSDR region) resulting in permanent stability of Foxp3 expression.